Protein phosphorylation is of great importance in growth factor action and control of the cell cycle. Numerous protooncogenes have been identified as tyrosine or serine/threonine or serine/threonine-specific protein kinases. Growth factor receptors recruit multiple Ser/Thr protein kinases in the execution of their cellular programs. Aberrant regulation of protein kinase function is often a pivotal element in cancer pathogenesis. An understanding of the structure, regulation and role of protein kinases in cellular growth control will help in design of pharmacologic agents a specific for particular kinases which may,, in turn, serve as useful treatments for cancer. I have recently detected and purified a novel rat liver protein kinase which is likely to be an important element in growth factor signalling. The enzyme, provisionally name as pp54 MAP-2 kinase, is a 54-kDa polypeptide which avidly phosphorylates microtubule-associated protein-2. The pp54 MAP- 2 kinase coelutes with one of the major peaks of mitogen-activated MAP-2 kinase activity. Moreover, the purified pp54 MAP-2 kinase appears to have been isolated in an "activated" form, in that it can be completely and specifically deactivated by treatment with the Ser/Thr phosphatase-2A, and independently by treatment with a recombinant phosphotyrosine phosphatase- 1B. The pp54 MAP-2 kinase is distinct in its substrate specificity from a p42 MAP-2 kinase described previously, however, both kinases exhibit dual regulation through Ser/Thr and Tyr-specific phosphorylation of the enzyme polypeptide, and constitute two members of a class of enzymes I have called "signal-integrating" protein kinases. This application aims to study three major aspects of pp54 MAP-2 kinase. 1) Regulation: These studies will attempt to identify Ser/Thr kinases capable of reactivating the phosphatase-inactivated pp54 MAP-2 kinase. Purified Ser/Thr and Tyr-specific kinase preparations will be assayed for reactivating activity as extracts from mitogen-treated cells. 2) Substrate specificity: Preliminary evidence suggests that pp54 MAP-2 kinases favors Ser/Thr residues with prolines located immediately carboxyterminal. Proteins rich in Ser/Thr-Pro motifs, specifically, RNA polymerase-II and transcription factors which have been implicated in growth regulation, such as p53 retinoblastoma gene product and c-abl, will be assessed as substrates of pp54 MAP-2 kinase. Potential substrates will be assayed not only for phosphorylation by pp54 MAP-2 kinase but, when feasible, for changes in function which are a consequence of phosphorylation. 3) cDNA Cloning and Expression: Structural analysis of pp54 MAP-2 kinase will be useful in relating this enzyme to other known kinases. Expression studies coupled with site-directed mutagenesis will serve to elucidate the role of pp54 MAP-2 kinase in cell function and will map enzyme function and regulation to particular domains.